The immune mechanisms that provide protection against disease and death due to malaria remain poorly understood. Furthermore, the immune responses that contribute to protective immunity elicited by active vaccination in animal models of malaria are also poorly defined. Although elucidation of the exact mechanism(s) of immunity is not a prerequisite to the development of efficacious vaccines, in the absence of reliable in vitro correlates of protective immunity and with the lack of some understanding of what constitutes clinical immunity, development of effective malaria vaccines will continue to be empiric and haphazard at best. The major goals of this project are to clearly define the basis of protective immunity that develops after vaccination with subunit asexual blood stage vaccines in experimental animal models and develop relevant biological assays that will assist in the evaluation of candidate asexual blood stage malaria vaccines in humans. We have now established that protective immunity can be elicited routinely by subunit vaccination with the merozoite Major Surface Protein 1 (MSP1) in both murine (Plasmodium yoelii in mice) and primate (P. falciparum in Aotus monkeys) malaria models. In the P. yoelii murine model several lines of evidence (e.g., passive transfer of antibodies or T-cells, vaccination of mu-chain knockout mice and studies of congenic mouse strains) consistently indicate that protective immunity is antibody-dependent; however, polyclonal antibodies alone, as assayed by ELISA, do not mediate protective immunity. Preliminary evidence suggests that the avidity and fine specificity of the polyclonal response contribute to protective immunity. A variety of in vitro assays and vaccination studies in genetically or pharmacologically manipulated mice have been undertaken to determine the immune mechanisms that contribute to antibody-dependent protection. Whenever feasible, comparable assays in the P. falciparum nonhuman primate model system have been pursued. The studies in primates, in which the use of Freund's adjuvant appears to be essential, have provided a basis for submission of an Investigational New Drug (IND) for testing of a MSP1-based subunit vaccine in Phase I human clinical trials. Freund's adjuvant has been the only formulation in the Aotus malaria model that has elicited detectable protective immunity. The role of Freund's adjuvant (which contains mycobacteria) is currently being investigated in studies of recombinant BCG. In addition, the contribution of cytokines, particularly IL12, in inducing protective immunity is actively being studied. Studies of alternative delivery systems/regimens, for example, priming with naked DNA injections and boosting with conventional subunit vaccination, is being studied in primates to determine if a formulation other than Freund's adjuvant can elicit protective immunity in primates.